Thousands of manmade chemicals have the potential to disrupt the endocrine system in humans. Termed endocrine disrupting chemicals (EDCs), they can cause negative effects on reproduction, metabolism and many other body systems. One potential source of human exposure to EDCs is through the use of hundreds of chemicals used in the process of hydraulic fracturing for natural gas and oil (fracking). More than 750 chemicals are reportedly used throughout this process, including more than 100 known or suspected endocrine disrupting chemicals. Spills and leaks of these chemicals and wastewater from the process are common. Importantly, the process was exempted from multiple federal regulatory acts in 2005 including the Safe Drinking Water Act, the Clean Water Act, and the Clean Air Act, which leaves regulation of this process largely with individual underfunded and understaffed states. Our overall hypothesis is that fracking and drilling operations contaminate surface and ground water with EDCs. We recently found an association between fracking and elevated endocrine disrupting activity in surface and ground water. Water from drilling dense areas had greater hormonal activity than drilling sparse reference sites in Garfield County. In the current proposal we aim to expand this analysis to 50 sites to determine if this association is broadly applicable and to identify chemicals responsible for the EDC activity. We will establish water quality measures and use forensic geochemical tools to determine if hydraulic fracturing fluids are present in the water samples. After solid-phase extracted, estrogen, androgen and thyroid receptor activity will be measured using reporter gene assays. We will use spatial analyses to determine if EDC activity is associated with proximity to drilling. Next, we will identfy endocrine active compounds by bioassay-guided fractionation followed by chemical profiling. It is essential to determine if fracking operations are contributing EDCs to water in order to determine the overall potential impacts of this process on human health. Completion of the proposed study will result in a more thorough understanding of the potential EDC contribution of fracking operations to surface and ground water in drilling-dense regions.